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Cloning PUFa [ PUF(3-2) ] and PUFb [ PUF(6-2/7-2) ] with N-terminal Nuclear Localization Signal (NLS) [1] were amplified from constructs containing these coding sequences with primers containing SgrAI and PacI sites and were used to replace SgrAI-dCas9-FseI from pAC164:pmax-dCas9Master_VP64 to create pAC1355:pmax-NLSPUFa_VP64 and pAC1356:pmax-NLSPUFb_VP64. A fusion PCR with 5’ fragment up to repeat 4 of NLSPUFb and 3’ fragment from repeat 5 to the end of NLSPUFa was used to create pAC1357:pmax-NLSPUFw_VP64. A fusion PCR of 5’ fragment of NLSPUFa with 3’ fragment of NLSPUb was used to to create pAC1358:pmax-NLSPUFc_VP64.

Casilio: a versatile CRISPR-Cas9-Pumilio hybrid for gene regulation and genomic labeling